Abstract (Project Summary/Abstract) Despite a strong and lengthy effort focused on developing an HIV vaccine, immune correlates necessary to achieve robust and sustained protection remain unknown. The discovery and characterization of numerous broadly neutralizing antibodies (bnAbs) that neutralize genetically diverse viruses, and the observation that bnAbs can passively protect against infection in nonhuman primates has generated optimism that strategically selected envelope (Env) immunogens will induce neutralizing antibodies (nAbs) that are broadly protective. Recent studies suggest that Env immunogens that (i) preserve features of the native trimer and (ii) are based on variants from individuals that developed bnAbs are worthy pursuits. We propose to utilize patient-informed Env immunogens delivered via DNA/modified vaccinia Ankara (MVA) immunization followed by protein boost to determine how the natural history and presentation of the Envs shape the genetic and functional antibody landscape in rhesus macaques (RM). The Env immunogen sets are derived from two HIV-1 infected individuals, chosen due to the disparate nature of their early antibody responses, which can be mimicked by vaccination, and subsequent development of ?elite? or ?poor? nAb breadth. Monoclonal antibodies derived from these two individuals 7 months after infection display striking differences in germline usage, clonality, binding affinity, and autologous neutralization. Thus, a detailed understanding of how antibody responses are influenced and altered by the immunogen choice and presentation form will ensure that vaccine efforts can be driven towards bnAbs, while simultaneously avoiding pathways that produce antibodies with limited function. DNA/MVA, a vaccine platform that produces robust and durable Env-specific antibody responses in RM and humans, and has provided protection against SIV challenge, will be used to deliver sequential patient-derived Env immunogens. These immunizations will be followed by a protein boost consisting of either gp120 or ?native flexibly linked? (NFL) gp140 stabilized trimers. Our ensuing multi-parametric analysis will include antibody germline usage, somatic hypermutation, development and longevity of clonal lineages, binding affinity (KD), and capacity to neutralize virus or facilitate Fc-mediated signaling, as well as post-immunization activation of germinal center (GC) B cells and T follicular helper cells (Tfh). To achieve an unprecedented level of resolution, our analysis will take place at the single B cell and monoclonal antibody level, examining plasmablasts, memory B cells, and long lived plasma cells residing in the bone marrow. Based on the results of these analyses, we will select individual B cells from immunized monkeys for RNA-Seq transcriptome analysis to connect B cell biology with antibody functional capacity. Our overall hypothesis is that the immunogens from the ?elite? neutralizer will elicit functional neutralizing antibody responses, and the trimer protein will further augment these. By contrast, immunogens from the ?poor? neutralizer will reveal mechanistic roadblocks to the desired antibody responses.